Our laboratory strives to understand the cooperative dynamics of proteins when they operate as highly organized and integrated assemblies. We adopt engineering approaches that enable model systems of natural multiprotein assemblies to be reconstructed in vitro while preserving the intricate molecular features of these assemblies. Our techniques allow the molecular orchestration of interacting proteins to be sensitively probed, while defining specific molecular interactions that are normally confounded by the diversity of cellular environments. In this way, we are able to bridge the gaps between our understanding of the stochastic dynamics of isolated single molecules and the operation of multiprotein assemblies in physiological settings. This research naturally involves a combination of state-of-the-art synthetic techniques to construct multiprotein architectures, and the development of instrumentation to investigate collective protein dynamics with single-molecule precision. While our methods can be generalized, enabling investigation of a variety of multiprotein networks and assemblies, so far this research concentrates on two areas:

IN VITRO MODELING OF INTRACELLULAR TRAFFICKING AND TRANSPORT:

The transport of intracellular cargo to different locations of the cell involves the activated mechanics of biomotor proteins. In many cases, these proteins interact with one another, as well as their associated regulatory proteins, in order to control and optimize transport. We seek to understand mechanistic pictures of these processes by reconstructing these assemblies in vitro and investigating their cooperative dynamics using single-molecule microscopy and spectroscopy techniques. Assemblies are constructed using engineered molecular scaffolds, the properties of which are defined by artificial proteins and DNA nanostructures. These scaffolds provide a means to accurately specify the supramolecular architecture of assemblies by defining the number and types of coupled motors, intermotor distances, and the elasticity of motor interconnects. These capabilities will enable us to develop detailed structure-activity relationships that will unveil the fundamental scaling laws that govern collective biomotor transport. Furthermore, we can probe the molecular orchestration that occurs within these assemblies with single-molecule sensitivity, bringing new insight to our understanding of the rich dynamics observed during motor driven intracellular transport.

ENCODING THE SELF-ORGANIZED MECHANICS OF INTERNALLY DRIVEN FILAMENTS:

The ability to generate coherent and sustained oscillations in highly viscous and damped cellular environments is of fundamental importance in biology. This type of mechanics provides cells with a means for self-propulsion and to stir their surrounding fluids using the controlled beating of internally driven filaments. Similar mechanical behavior is harnessed by cells to detect and amplify extremely small signals, which in some cases, are no larger than thermal noise. Motions of these filaments are self-generated by large collections of biomotor proteins that are arranged into complex architectures, such as those seen in axonemal cilia and flagella. With these systems as inspiration, we are engineering model biomotor assemblies that operate as shearing-mode oscillators, and emulate the dynamics of axonemal assemblies. Using artificial proteins to define and tune the properties of biomotors and nanomechanical devices to probe the motions of these motors, we are developing strategies to encode the cooperative dynamics of internally driven filaments. These efforts will impact our fundamental knowledge of collective biomotor mechanics, and provide guidelines to harness the mechanochemical mechanisms cells employ to sense, respond, and adapt to their surroundings.

Department of Molecular Physiology and Biophysics, University of Vermont Medical School (September 2015): “Synthetic manipulation and analyses of cytoskeletal transport and structural regulatory proteins”.